Chaperones - Science topic

I assume you are purifying His-tagged protein by using Ni2+ or Co2+ based resin, then it may be helpful to wash the column with your wash buffer containing additional 2.5 mM ATP, 5 mM MgCl2, and 0.5% Triton X-100 or NP-40. By doing this, you may remove the co-bound Cpn60 or DnaK protein.

A possibility is that it is the same protein that is longer because of read-through of the stop codon until a second stop codon was reached. You could check this by doing a bit of sequencing.

how have you evaluated the expression? Western blot?

it is possible that your western blot had an issue. Sometimes it’s easy to mess up your western blot samples and have the proteins degrade.

I suggest:

1. As a control, transfect mammalian cells with the plasmid for this protein. Get the cell lysates from that. Retransform your yeast (just redo it all). Get the cell lysates from that. Now you have cell lysates from

1. Untransfected mammalian cells

2. Transfected mammalian cells

3. Untransformed yeast

4. Transformed yeast.

run the western blot with these. See if you are getting a band for just the transfected mammalian cells. Then you know it’s a yeast producing the protein issue, maybe a chaperone expression issue. If you get a hand for both, great. If you get a band for neither:

you need cell lysates from cells that express the protein natively. This is the best positive control.

run western with that and see if you get a band: if you do, then you know that it’s an issue with the plasmid. If you don’t, then it’s an antibody issue.

Youve got this. Best of luck. It’s going to all work out!

1. Improve cell culture and expression conditions to reduce the amount of heat shock proteins produced. 2. Use affinity chromatography to purify the monoclonal antibody, as heat shock proteins have a high affinity for certain resins which can be used to separate them from the desired product. 3. Utilize size exclusion chromatography to remove low molecular weight heat shock proteins that can't be removed by affinity chromatography. 4. Utilize ion exchange chromatography, as this technique can be used to separate heat shock proteins based on their isoelectric point. 5. Utilize hydrophobic interaction chromatography, as this technique can be used to separate heat shock proteins based on their hydrophobicity. 6. Use a combination of various chromatographic techniques to maximize the removal of heat shock proteins from the desired product.

Hello Pushpender Bhardwaj

You may use the Lyso-IP method. With the Lyso-IP technique, intact lysosomes are immunoprecipitated via epitopes that are added to the cytosolic portion of lysosomal transmembrane proteins. The fusion of triple human influenza virus hemagglutinin (3×HA) epitope tags to the lysosomal transmembrane protein 192 (TMEM192), TMEM192-3×HA, allow for the rapid isolation of lysosomes by using anti-HA magnetic beads. Remarkably, this takes only approximately ten minutes to accomplish. The isolated lysosomes can retain the lysosomal protease activity and also contain many lysosomal luminal proteins.

Additionally, the rapid timeframe of the TMEM192-tagged Lyso-IP method allows potentially labile molecules, such as the amino acids and other metabolites to remain stable for downstream analysis. Lysosomes isolated by the Lyso-IP method can be successfully used for a number of analysis such as proteomic, lipidomic, and metabolomic analysis.

Please refer to the references below.

1. https://www.science.org/doi/abs/10.1126/science.aan6298

2. https://pubs.acs.org/doi/abs/10.1021/acs.jproteome.9b00580

Best.

Dear Nida Y.Mah

Sumo as MBP as good solubility and may improve the solubiliy of some proteins but it depends a lot from the protien properties and from the reason why the protein is insobule (membrane protein, unfolded, aggregate, toxic for cells)

Did your protein contain hydrofoic regions or many cisteines?

best regards

Manuele

There are many examples of proteins and pathways that serve multiple roles. So it is certainly possible that the T3SS has multiple roles which include both stress response and virulence and these activities may be regulated differently as well. There is no real answer to which is the "true" role.

Chaperones bind to hydrophobic stretches of the nascent polypeptide chain, shielding these surfaces till a full domain or the full polypeptide chain is released from the ribosome. Chaperones also reduce "polypeptide-chain"-"polypeptide chain" interaction in a crowded environment and consequently protein aggregation.

Its mechanism of action, depending on or not of ATP, must provide an exposed hydrophobic surface. Thus, it is likely that when chaperones are expressed in high amounts may aggregate forming including bodies.

Thank you so much it worked out

Thanks for the answers, the construct is just a simple T7 expression vector (lac operon), there's no signal peptide. It would have been nice to get a single band but I may have to push on with purification without it as we need a high yield. But I will definitely take a look at a periplasmic extraction.

Pratibha Srivastava Thank you！

Hi Irene Feliciotti

You may find the following pdf helpful.

Did you do the codon optimization for the expression MMLV-RT in E.Coli?

Be careful with TCEP.

It does have a mild cysteine directed side-reaction proteolytic activity that may be a problem in some cases.

Ref:

https://doi.org/10.1016/j.jasms.2010.01.016

Title:

A Tris (2-Carboxyethyl) Phosphine (TCEP) Related Cleavage on Cysteine-Containing Proteins

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon–carbon bonds in cysteine.

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Hi guys,

I found the sequence here:

https://www.lifescience-market.com/plasmid-c-94/pgro7-plasmid-p-63479.html

Hi Felix,

I would say that co-expression of chaperones in my experience has definitely worked in the past but choosing the best fit is important. If you can afford investigation of a few different chaperones using literature info. this is the way to go. Just a couple of examples:

This review also highlights some good references of when chaperones have and haven't worked (good source of more literature): << the review also lists some "expression kits" which could be useful - not tried them personally.

As you will find, there is a lot of context dependency in the literature, what has worked for others and no guarantee that it will work for your system. Then there are additional considerations of expression levels of the chaperones, when and how they are induced - so you can immidately start seeing that this is a multi-factorial problem. I would say a great opportunity to think about start using DoE to explore (happy to share more here if interested).

Just some additional thoughts:

Are you proteins definitely stressful for your cell? I imagine you know this from recombinant versus non-recombinant Pichia comparison data? For example, you can see you've got reduced biomass. Typically, recombinant expression and growth are perpendicular, although there's interesting literature where the balance can be fine tuned (give continuous fermentation processes or bioprocesses a Google).

If you can, it would be worth identifying which one of the two proteins is problematic, if possible. It is also worth asking the question of what your end goal is? Are you developing a pathway? Are you expressing a biologic? etc. Depending on this you may use a different approach.

Also, how much do you know about the proteins? Is the structure known, do they contain disulfide bonds (i imagine so, if you've gone for PDI)? Is there a requirements for cis-trans propyl isomerase? etc.

Additionally, think about how you're currently expressing these proteins as well as there are a lot of control elements from the promoter you use, to its transcription initiation rates, RBS strengths, etc. that can help you achieve similar results. You may find that one of the proteins is expressed at too fast a rate, etc. Here's an example of how you might want to modularise your expression system to investigate further. https://www.addgene.org/kits/sieber-moclo-pichia-toolkit/

It's could also be useful to think of how you could modulate the expression of secretion machinery, even change the secretion tag which could be having an impact on expression levels.

Hope this was useful - but there's definitely a lot of information out in the literature.

Best of luck with your experiments!

Tagging or measuring the levels of a known autophagy substrate is one of the most robust ways to measure autophagy.

Fluorescent protein based tags like Keima and Rosella allow you to quantify and measure the delivery of your substrate to a lysosome. For CMA, you would need to put the tag on an established CMA substrate like GAPDH, or something else with the KFERQ-like motif in their sequence. Alternatively, you can immunoblot for the total levels of those substrates (as long as you exclude the role of other autophagy pathways in their clearance). hope that helps

Hi there,

You can use Tris/HCl-based or even HEPES-based buffers for His-tag purifications, there is no need for a phosphate buffer.

As an alternative, you could dialyze your POI to remove the phosphate and change to a HEPES- or Tris-buffer, before treating the protein with Mg/ATP.

We have only limited experience with Mg/ATP treatment, but it did not work out very well. When the chaperones were removed, the proteins just aggregated. So there seems to be a good reason (not stabilly folded proteins), when chaperones bind to your protein to keep it soluble. It might, therefore, make sense to invest time in the optimization of expression conditions.

Good luck,

Sebastian

False positives are pretty abundant (more than 50%) when the conventional Yeast Two Hybrid assay is used.

The next generation of Y2H system, called the NGIS, overcomes the false positives, and gives you precise hits. Its also more comprehensive, gives you 10-100x more hits and the weak transient PPIs can also be detected. Much less time and cost for downstream confirmation assays.

For advice ask Bernie from Next Interactions.

Agree with Laura: polyclonal would be informative: are all three bits from the same protein?

But theoretically, all 3 bits should have the his tag, and anti-his Ab might be helpful too.

If the bits are indeed the same protein, the PMFS might be "off" and protease/s are chewing off the carboxy terminal (assuming its the amino terminal that has the his tag!). We always use leupeptin and PMSF, and even p-complete protease inhibitor cocktail tablets!.

Hi Dilshan, one problem that I foresee is that many intracellular misfolded proteins are ubiquinated and degraded by proteasomes.

I got the sequence from Steffen's link. Thank you !

Dear Colleagues,

I would like to share our findings regarding camel somatic cell thermotolerance to 45 C for 20h.

DEar ZAc

but the his tag is at the N term or at C term?

You can certanilly as Sebastian told you add an N-terminal fusion patner (eg. GST, GSt, Trx, GB1, ZZ) and a enzime restriction site (i prefer TEV to thrombin) that will allow to you to digest your protein and remove the tag after puriication. pmal, pgex are some example of commercial vectors that may allow to you to build this construct). pETG vectors (https://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/gateway_vectors/) are an interesting set of vectors developed from EMBL that may allow to you to test more tag in parallel. As accademinc reseacher I had the opportunity to use it many years ago during my PhD but i'm not sure if is possible to obtain it. However with modern enzime free cloning approach as PIPE metohds is possibile to modify directly your expression vector buy the addiction of a small tag as Trx.

good luck

Manuele

thank you.

Hi there,

I'm sure that there are a lot of possible approaches to screen peptide libraries for peptides that prevent A-beta aggregation in-vitro.

However, in contrast to small molecule inhibitors, the main issue with peptides will be the delivery: It will probably not be easy for your peptides to cross the blood-brain-barrier and get into the central nervous system.

Another important aspect is the in-vivo stability of your peptides. If they are unstable and rapidly degraded in-vivo, they will not be very useful. At the same time, you want to make sure your peptides cannot oligomerize or aggregate.

You will find a bit about analytical methods for the characterization of therapeutic peptides here:

Best,

Sebastian

Hi there,

5 mM ATP/Mg in washing buffer should do the job.

I would say clathrin coated vesicle uncoating.

Therefore the formed vesicles remain entrapped in their clathrin lattice and cannot fuse.

Contact me I can give you more details.

e

Pilot screening of protein expression in presence of different concentration of ethanol

 

1.

Pick a single colony from the transformed LB agar plate or take 20 μL of glycerol stock and inoculate into 5 mL of LB broth containing appropriate antibiotics according to the vector construct and host cells.

2.

Incubate the culture at 37 °C for overnight (14–16 h) at 180 rpm with continuous shaking as starter cultures.

3.

Next day take five culture vials and name them as:

•

Control (un-induced).

•

Induced in the absence of ethanol.

•

Induced in the presence of 1% ethanol.

•

Induced in the presence of 2% ethanol.

•

Induced in the presence of 3% ethanol.

 

4.

Inoculate five vials (each containing 5 mL LB medium with appropriate antibiotics) with 20 μL of the overnight grown starter cultures.

5.

At the time of inoculation add 1%, 2% and 3% ethanol to the LB medium and grow at 37 °C for a few hours (approx. 3–4 h.) with vigorous shaking, until the OD600 reaches 0.5–0.6.

Note: Ethanol should be used in v/v ratio. We have optimized the optimum ethanol concentration and it was observed that 3% ethanol (v/v) gives the maximum enhancement in protein expression. In the presence of more than >5% ethanol cell growth was found to be inhibited.

6.

Induce the protein expression by adding IPTG to a final concentration of 1.0 mM.

Note:

•

Do not add IPTG to the culture which will serve as a non-induced control.

•

Minimal IPTG concentration should be optimized in small scale of culture before proceeding to the mass culture.

•

The optimal growth time for TB (Terrific broth) is different from LB (Luria broth) In case of TB OD600 should be more than 1.0–1.5 before IPTG induction.

•

In case of auto-induction media the control should be normal LB not the auto-induction media. There is no need to observe the OD, because it does not need the IPTG induction.

 

7.

Grow the cultures for an additional 4–5 h at 37 °C with continuous shaking.

8.

Harvest 1 mL of cells from each culture vial by centrifugation for 1 min at 12,000 rpm. Discard the supernatants (remaining media).

9.

Re-suspend the cells in 60 μL of buffer (Tris or phosphate or PBS, pH-8.0), 20 μL of 10% SDS & 20 μL of 5X SDS loading dye and lyse by mixing or pipetting.

10.

Boil the samples in 100 °C for 5–10 min (Vortex the samples in between).

11.

Load the sample in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and analyze. Screening through pilot experiment is important, which will give the clear picture about the level of over-expression of the protein in the presence of 1%, 2% and 3% ethanol. This will also provide the opportunity to compare the over expression of recombinant protein in the presence and absence of ethanol. SDS-PAGE analysis will support the increase fold of expression with increased percentage of ethanol.

 

Note: Pilot screening can be done at low temperature ranging from 16 °C to 23 °C for 20–22 h after induction with IPTG, depending upon the level of expression and solubility screening of the recombinant proteins. In case of auto-induction media incubate the culture until the OD600 reach 0.4–0.6. It is observed that some proteins give higher fold of expression in low temperature. This screening can also provide a clear idea about the level of expression of the protein, which can be helpful for further mass culture. For mass culture setup the experiment according to the same ratio of pilot experiment.

Thank you for that article Kristyna. There are some ideas which I want apply in my assays. 

welcome

Instead of freeze-drying, why don't you just flash-freeze the purified protein in the liquid state in small aliquots and store it at -80 C? This avoids excessively high salt in the samples. If the protein is too dilute, use ultrafiltration to concentrate it before freezing.

yes you can eliminate non-specific interactions during coimmunoprecipitation by giving extensive  buffer washes but it also eliminates weak binders and try to use negative control where you can compare proteomes that you have resolved on 2D gels of both your control and test experiment.you can subtract common proteins that were precipitated in control and your test

Thank you! 

Try another coupled TTS, this one may be depleted few rare tRNAs. Alternatively redesign your template using exactly identical codons for both proteins.

Thank you for your answers and words of encouragement. 

I had similar problem and the protocol of Rohman and Harrison-Lavoie (Separation of copurifying GroEL from glutathione-S-transferase fusion proteins. Protein Expr. Purif. 2000, 20: 45-47) worked perfectly. Briefly, you can denature by heat lysate of uninduced bacteria and add it to your lysate of interest. You should simultanously add ATP and MgCl2 and incubate shortly in 37 C degrees. Chaperones should bind to denatured protein which was added in excess. Then you can continue with purification procedure.

Hi,

As Akash mentioned the chaperone might be binding to your proteins. Adding ATP and MgCl2 can help in getting rid of the bound chaperone. You may have tried this already. If this works your original two proteins should still be in a proper complex. Do a careful gel filtration to rule out the presence of soluble aggregates. You can do the gel filtration with your original complex too. A single well resolved peak of the complex on gel filtration will go a long way in furthering your project.

Good luck

Hi Patricia,

Every protein is different hence you may need to trouble-shoot expression issues. There are a few things to take in to consideration (I'm assuming that the protein is foreign to E. coli?)

1. Try different growth conditions: i.e. induction time length (sample over a number of hours post induction), temperature (lower temp helps with protein folding), media (try different media: 2xYT, LB, Teriffic broth). TB is pH buffered and may help with expression in some cases.

2. Plasmid/promoter: T7 is a powerful promoter and may not be suitable for expression of all proteins. Use a weaker promoter, we had excellent results with pTrc (invitrogen) for soluble protien.

3. Even try a different strain of bacteria.

4. As suggested by Garitsa you could try periplasmic expression

5. You could try a codon biased strain i.e. RIPL

good luck

Is the expressed protein soluble/useful? It does not matter if the chaperone is expressed or not. If it is not soluble (I assume that is the case) here are a few things that you can think of:

1.How are you looking for the soluble protein? Can you pick it up using a western blot? The SDS-PAGE comassie may not show a good thick band.

2. Do you have an assay for this protein? (is it an enzyme?)

3. See PMID:19074766 for a test case. The takara chaperone system works only sporadically and many proteins are still unstable and the increase in yield is minimal. Make sure your lysis buffer has a variety of protease inhibitors.

4. Do a careful time vs inducer (Tet/arabinose, or whatever) profile at various cell densities(0.5, 0.8 and 1.2 OD600). If you are lucky there might be a condition that is optimal but not by much. This may be good enough for your purpose. Induction temperature is another variable that you might add to the mix. It is quite a bit of work right there.

Good luck

Dear Papadopoulos， Thank you very much for your answer, I will consider it according your suggestion.

Yes, chaperones bind very strongly to substrates, because the extraction buffer dilutes any ATP that might be present in the cells, and when the first precipitation and wash is done, there is no more ATP left and the ADP-bound form of the chaperone binds stronger than the ATP-bound form. This means the extraction buffer can have detergents, chaperon-substrate interaction studies are amongst the easiest IP models you can find, at least this is true for the HSP70 family of proteins.

A typical homogenization buffer is 200 mM Tris-Cl, pH 8.0, 300 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. The high salt helps to solubilise large protein complexes

As binding buffer you can use NET gel buffer (50 mM Tris-Cl, pH 7.5, 150 mM; NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and 0.02% NaN3 and supplemented with 0.25% gelatin). The detergent increases the specificity of the IP, most non-specific protein-protein interactions are broken in this buffer but the chaperone keeps binding.

To test ATP-dependent release, you can resuspend the washed ProteinA-sepharose pellet in release buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 3mM ATP and 6 mM MgCl2), incubate for an hour, then discard the pellet and re-immunoprecipitate with antibodies to the test substrate.

A typical protocol for in vivo labelling with 35S methionine and details of the amounts used for IP and ATP-release + re-IP can be found in the following reference:

Goof luck :) 

You might like to take a look at 

TAT-mediated protein transduction into mammalian cells.

Becker-Hapak M, McAllister SS, Dowdy SF.

Methods. 2001 Jul;24(3):247-56. 

PMID: 11403574

The authors intentionally denature their TAT-fused proteins by solubilisation in urea followed by rapid removal of the urea. In some cases they found this increased the biological effects of the proteins on cells - they speculate this facilitates entry into the cells. These findings suggest that at least these proteins, once in the cells, can refold to an active conformation.

I dont get complete inhibition. all i am saying is that whatever percent of inhibition i am getting is, above one molar ratio, invariant. So, say, 1:5 and 1:10 the graph is the same. Aneways, thank you all. I got my answer from Mr. Jogender Singh. Thank you again.

@ Soren - Yes i need the protein. Can I get the antibody for that protein?. Please provide the details. I will send a request for your lab. Thanks a lot.

The problem with the use of nanobodies might be that they are conformation-specific. To my knowledge, nanobodies are still only obtained by injecting the protein into live animals (lamas, typically, unless you have a pet shark). If your protein has various conformations, you'd need to stablise a particular conformation prior to injecting into the animal, to obtain the appropriate nanobody. If your protein region of interest is intrinsically disordered, then this might not work.

So you might be better off choosing a natural ligand for your flexible part, and then crystallise. 

The most important question would be why you wish to obtain a crystal structure of an unstructured region? What would be the biological insights gained from such a structure? As an alternative, you could use SAXS or NMR to characterise this region in its unliganded state. If you have biological ligands, then you could add those to SAXS / NMR and analyse. And then only crystallise a relevant fragment bound to  3D ligand.

Based on our previous observations, a combination of osmolytes (25% glycerol, 500 mM Sucrose and ATP in the presence of Mg+2 and K+ at 30oC (chaperonins function better at higher temperatures,) was used by a collaborator to remove GroEL from binding to the difficult to purify protein, removed GroEL as assessed by SDS PAGE and used this protein to acquire NMR structural data.    This combination may actually help fold the protein on column (we have observed this with multiple proteins).  This particular protocol called for a slow flow rate of 0.2 ml/min with 5 column volumes of 100mM Tris (pH 7.5 (not buffered well but Tris works well to help fold proteins), 100 mM KCl (GroEL is a K+ ATPase), 25% glycerol, 500 mM Sucrose, 20 mM MgCl2, 5 mM ATP, 0,5 mM PMSF (may not be necessary if only two proteins are GroEL and your protein of interest), 1mM DTT (if you need to maintain reduced Cysteine).  Your wash buffer should contain GroEL.   Alternatively, if you are having problems in acquiring natively folded protein, than you might want to actually consider ADDING chaperonin to the column to facilitate on column folding. see some references below.  The collaborator never published this paper for some unknown reason (was not science related).....So if you use this method, reference the pubs below.... thanks and hope this helps.

Katayama H., McGill M., Kearns A., Brzozowski M., Degner N., Harnett B., Kornilayev B., Matkovic-Calogovic D., Holyoak T., Calvet J.P., Gogol E.P., Seed J., and Fisher MT., (2009) “Strategies for folding of affinity tagged proteins using GroEL and osmolytes” J. Structural and Functional Genomics. 10, 57-66. epub Dec 2008

Voziyan P.A. Jadhav, L and Fisher M.T. (2000) “ Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of chaperonins and osmolytes.” The Journal of Pharmaceutical Sciences 89, 1036-1045.

It might not be absorbance you are observing but light-scattering. The GA may be starting to crash out. If this is the case, then changing the excitation/emission wavelength will not help. Lower the concentration of GA, increase DMSO or try more soluble version of GA.

Topology of energy landscape for proteins is very complex. Imagine a system with N atoms, then every configuration of this system in phase space is characterized by 3N degrees of freedom. In particular, macromolecules are characterized by multiple minima energy landscape because there exist many possible conformations, which are separated by high barriers dominated by both energetic and entropic contributions.

To parametrize this energy landscape is very difficult, often what is done, is determining a few generalized coordinates, sometimes called collective coordinates, which can characterize most of the fluctuations on the system, then using particular efficient sampling techniques to characterize changes on the energy landscape along only these reduced degrees of freedom.

The most used sampling techniques include, ombrella sampling, transition path, metadynamics, targeted MD, and maybe others.

I would suggest two proteins – α-synuclein and LRRK could be taken as the two major proteins to be targeted for PD; and one of the starting articles could be the following recent review:

Breydo L, Wu JW, Uversky VN. Α-synuclein misfolding and Parkinson's disease. Biochim Biophys Acta. 1822(2):261-85, 2012. A copy of the paper could be received from the author: vuversky@health.usf.edu

Co-chaperones may be loosely defined as proteins that participate in the function of other chaperones.Co-chaperones provide a method for the direct recruitment of different client proteins to chaperones. Molecular chaperones are proteins that recognize and bind to other (partially) unfolded proteins or exposed stretches within proteins. By binding to the unfolded/misfolded substrates, molecular chaperones prevent aggregation and hereby support protein (re)folding; when refolding fails (e.g. in case of genetic mutations), the same chaperone action ensures that the client remains soluble and can be degraded by the proteasome or via chaperone-mediated autophagy (CMA). If these actions fail and (mini)-aggregates arise, the cells can use macroautophagy. In addition some chaperones, like e.g. HSPB1, may regulate the apoptotic process.

References

1. Cell Stress Chaperones. 2003 April; 8(2): 105–107.

2. Progress in Neurobiology 97 (2012) 83–100

3. Quarterly Reviews of Biophysics 44, 2 (2011), pp. 229–255